Green fluorescent protein (GFP) derived from Aequorea victoria, a jellyfish, has many purposes in biological systems. Recently, various GFP mutants have been produced based on the random mutagenesis and semi-rational mutagenesis, wherein a color is changed, a folding property is improved, luminance is enhanced, or pH sensitivity is modified. Fluorescent proteins such as GFP are fused with other proteins by gene recombinant technique, and monitoring of the expression and transportation of the fusion proteins is carried out.
One of the most commonly used types of GFP mutant is Yellow fluorescent protein (YFP). Among Aequorea-derived GFP mutants, YFP exhibits the fluorescence with the longest wavelength. The values ε and Φ of the majority of YEPs are 60,000 to 100,000 M−1cm−1 and 0.6 to 0.8, respectively (Tsien, R. Y. (1998). Ann. Rev. Biochem. 67, 509-544). These values are comparable to those of the general fluorescent group (fluorescein, rhodamine, etc.). Accordingly, improvement of the absolute-luminance of YFP is nearly approaching its limit.
In addition, cyan fluorescent protein (CFP) is another example of the GFP mutant. Of this type of protein, ECFP (enhanced cyan fluorescent protein) has been known. Moreover, red fluorescent protein (RFP) has been isolated from sea anemone (Discoma sp.). Of this type of protein, DasRed has been known. Thus, 4 types of fluorescent proteins, that are, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and red fluorescent protein, have successively been developed. The range of the spectrum has significantly been expanded.
Previously, the present inventors had succeeded in amplifying a fluorescent protein gene from among the cDNA library of Fungia sp., using preferred primers designed based on the amino acid sequence of a known fluorescent protein, and then cloning it. Thereafter, the present inventors had determined the fluorescence properties of the obtained fluorescent protein derived from Fungia sp. As a result, the present inventors had found that the above fluorescent protein has desired fluorescence properties (International Publication WO03/54191).
Several GFP homologs derived from Aequorea have a large stokes shift (the difference between an excitation peak value and a fluorescence peak value) (GFPuv and sapphire). These GFP homologs obtain green fluorescence as a result of excitation with UV light at 380 nm. However, the use of such UV light having toxicity is not suitable for observation in organisms. No red fluorescent proteins have a large stokes shift. Under the current circumstances, either excitation or fluorescence must be sacrificed in fluorescence observation.